Effect of FGF21 on the mitochondria respiratory capacity and mitochondria number in human dopaminergic neurons. Human dopaminergic neurons were stimulated with 50 ng/ml FGF21 and analyzed further as indicated below. (A) Oxygen consumption rate (OCR) in human dopaminergic neurons was measured in real-time using the Seahorse equipment as described in Methods. Typical graphs after addition of the various inhibitors are shown. (B) Quantification. Basal and maximum respiration (OCR) were increase in cells treated with FGF21 as was the spare respiratory capacity. Values are means ± SD, n = 4. ***p < 0.001 for FGF21 vs C. (C) Immunoblots. β-actin was used as control. The mitochondrial proteins TFAM and COX IV showed no significant changed in dopaminergic neurons treated with FGF21. (D) The mitochondrial DNA (mtDNA) copy number was analyzed using quantitative PCR as described in Methods. There was no change in the relative ratio of mtDNA to nuclear DNA reflecting an equal number of mitochondria in control and FGF21-treated dopaminergic cells. (E-F) Mitochondrial surface area. Left, control and FGF21 treated cells were analyzed by electron microscopy and the relative mitochondrial area was calculated from EM pictures as described in Methods. There were no significant changes in the mitochondrial surface area between control and FGF21 treated cells. Values are means means ± SD, n = 4. Right, typical EM pictures of control and FGF treated cells.